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The detection of novel or re-emergent pathogens necessitates the development of rapid, easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Heartland virus (HRTV) is the first pathogenic Phlebovirus responsible for serious and fatal cases in the United States. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of HRTV. The RPA-LF detected HRTV with a limit of detection of 1.19-1.54 plaque-forming unit equivalents/reaction. In addition, the RPA-LF was able to detect 0.6075 copies/μL of HRTV nucleoprotein gene-containing plasmid. We evaluated six clinical samples that were previously found to be real-time PCR positive for HRTV and found five out of six samples to be positive by RPA-LF, yielding 83.3% concordance with real-time PCR. All six samples had Ct values between 29 and 39 by real-time PCR. We also determined that the HRTV primers and probe do not cross-react with other tick-transmitted viruses such as Bourbon and Powassan, or other related viruses, including Lonestar tick virus and Sunday canyon virus (100% specificity). This is the first isothermal amplification test developed for a tick-borne virus, which will allow for rapid differentiation between HRTV and other pathogens producing similar clinical manifestations.

Chagas disease is a lingering Public Health problem in Latin America with ∼5.7 million people infected with Trypanosoma cruzi. Transmission is still taking place in most countries of the Americas, including the United States. Dogs are frequently infected with T. cruzi and its high infection prevalence is associated with increased risk of Chagas disease in humans. The city of Mérida in the Yucatan peninsula is endemic for Chagas disease and canines are frequently infected with T. cruzi. The objective of this study was to evaluate the performance of a qualitative point of care (POC) molecular test (RPA-LF, recombinase polymerase amplification-lateral flow) developed in our laboratory for identifying infected dogs. We used retrospective samples of dogs that came for consultation because of cardiac alterations and proved to be infected with T. cruzi as determined by enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative PCR (qPCR). The analytical sensitivity indicated that RPA-LF amplified T. cruzi DNA in samples containing almost equal to one to two parasites per reaction. Serial twofold dilutions of T. cruzi epimastigotes showed that the test had 95% (19/20) repeatability at concentrations of two parasites per reaction. The test showed no cross reactivity with human DNA or other protozoan parasites (Trypanosoma rangeli, Leishmania spp., and Plasmodium spp.). RPA-LF had the capacity to amplify all discrete typing units (DTUs I-VI) of T. cruzi that circulate in domestic or extradomestic environments. The RPA-LF had 93.2% (95% confidence interval 87.2-98.1) sensitivity and excellent agreement with qPCR used as gold standard (Cohen's Kappa test = 0.963). ELISA was positive in 96.6% (85/88) of dogs, which together with the molecular tests confirmed the frequent contact with infected triatomine bugs in the city of Mérida. These preliminary results on the diagnostic efficacy of the RPA-LF deserve further large-scale field testing of this POC test for T. cruzi infection in endemic areas.